A method for making a porous scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal

ABSTRACT

A method for making a porous devitalised scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal comprises the steps of providing micronized extracellular matrix (ECM) tissue, mixing the micronized extracellular matrix with a liquid to provide a slurry, and freeze-drying the slurry to provide the porous scaffold. A porous scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal and comprising a porous freeze-dried matrix formed from micronised decellularised extracellular matrix tissue is also described.

INTRODUCTION

The invention relates to a method for making a porous scaffold suitablefor use in repair of osseous, chondral, or osteochondral defects in amammal. The invention also relates to a porous scaffold suitable for usein repair of osseous, chondral, or osteochondral defects in a mammal,and a multilayer scaffold suitable for use in repair of osteochondraldefects in a mammal.

In humans, 95% of defects to the articular surface of synovial jointsinvolve cartilage without affecting the subchondral bone (Hjelle et al.,2002). Such defects fail to heal spontaneously. An estimated 5.4 millionpatients in the US alone will require joint and cartilage procedures totreat such defects and other degenerative changes by 2019. Bone marrowstimulation techniques such as microfracture are the most readilyavailable clinical repair strategies for articular cartilage (Getgood etal., 2009). By surgically penetrating the subchondral bone, progenitorcells from the bone marrow can migrate into the defect and form a repairtissue. In general, a mechanically inferior fibro-cartilaginous tissueis produced which provides only temporary symptomatic relief.Alternative cell based therapies such as autologous chondrocytesimplantation (ACI) are available, however these approaches require twohospital stays and are very expensive (˜

35,000), which may explain their relatively limited clinical uptakecompared to marrow stimulation techniques. There is therefore asignificant commercial opportunity for a cost effective ‘single-stage’or ‘in-theatre’ therapy (such as the proposed scaffold) for regeneratingdamaged articular cartilage.

Scaffolds fabricated using decellularized extracellular matrix (ECM)have shown great promise for the regeneration of damaged tissues. Thisapproach has been used to develop different tissue-specific (e.g. heartvalves, blood vessels, skin and cartilage) scaffolds. In the case ofarticular cartilage, numerous studies have demonstrated that scaffoldsderived from devitalized cartilage are chondroinductive and show greatpromise for regenerating damaged joints. There are, however, a number oflimitations associated with current ECM derived scaffolds, includinginhomogeneous pore size with current cartilage ECM derived scaffolds(which limits cellular infiltration into the scaffold and leads toinhomogenous deposition of matrix within the scaffold), failure togenerate hyaline cartilage within the scaffold or a defect treated withthe scaffold, poor control over scaffold pore size, inefficientdecellularization of ECM prior to scaffold fabrication, variability inscaffold composition which impacts commercial production, and poorcontrol of the release of exogenous growth factors loaded onto ECMderived scaffolds. Furthermore, it remains unclear how ECM derivedscaffolds can be used to treat defects that effect multiple differenttissues such as osteochondral defects”.

It is an object of the invention to overcome at least one of theabove-referenced problems.

STATEMENTS OF INVENTION

The Applicant has discovered that freeze-drying a slurry of micronizedextracellular matrix (ECM) tissue derived from hyaline cartilage(preferably articular cartilage) or growth plate tissue providesscaffolds having a homogenous pore size (FIG. 1) that demonstrate a highlevel of homogenous stem cell infiltration in-vitro (FIG. 2) and a highlevel of deposition of cartilage-like extracellular matrix (FIG. 3). TheApplicant has successfully employed decellularization techniques inorder to sufficiently reduce xenogeneic DNA from the scaffolds (FIG. 8).

Cartilage extracellular matrix consists primarily of glycosaminoglycans(GAG) and type II collagen. The Applicant has discovered that byreducing the glycosaminoglycan content of the cartilage ECM using adetergent or similar (FIG. 8), and hence increasing the ratio ofcollagen to GAG within the treated ECM, improves the resultant capacityof the scaffold to induce robust chondrogenesis after they have beenseeded with mesenchymal stem cells (FIGS. 9 and 10). The Applicant hasalso discovered that crosslinking the scaffolds of the invention slowsgrowth factor release from the scaffolds (FIG. 4), and that reducing theglycosaminoglycan content of the scaffolds of the invention also slowsgrowth factor release from the scaffold (FIG. 7). The Applicant has alsodiscovered that scaffolds formed from micronized growth plate ECMgenerates extensive mineralisation of cranial and femoral defects (FIGS.23 and 24) and enhanced bone tissue formation (FIG. 25), and that growthplate ECM derived scaffolds that are seeded with mesenchymal stem cellssupport endochondral bone formation in chondrogenic conditions in vitro(FIGS. 19-21).

The Applicant has also discovered that treated or native ECM can besolubilised by solubilisation of the ECM. After solubilisation the ECMcan then be cross-linked and freeze-dried to create scaffolds. Thesolubilisation process employed removes the vast majority of GAG andresidual xenogeneic DNA from the resulting scaffold. (FIG. 25).

Accordingly, in a first aspect, the invention provides a method formaking a porous scaffold suitable for use in repair of osseous,chondral, or osteochondral defects in a mammal, the method comprisingthe step of:

-   -   providing a slurry of micronized extracellular matrix (ECM)        tissue or a gel comprising solubilised and crosslinked        extracellular matrix (ECM) tissue; and    -   freeze-drying the slurry or gel to provide the porous scaffold.

Thus, the ECM material that is freeze-dried may be a slurry formed frommicronized ECM or it may be a gel formed by solubilisation of ECM(optionally micronized ECM) that is cross-linked, typically chemicallycross-linked, to form a gel prior to freeze-drying. Preferably, thesolution of enzymatically digested ECM is cross-linked prior tofreeze-drying.

In one embodiment, the ECM is solubilised by enzymatic digestion. In oneembodiment, the ECM is micronized prior to solubilisation.

Preferably, the slurry comprises 100-400 mg/ml micronised ECM tissue,ideally 200-300 mg/ml micronised ECM tissue.

Preferably, the micronized extracellular matrix tissue has a meanparticle size of 10-200 microns, ideally 20-70 microns.

Typically, the micronized extracellular matrix tissue is cryomilledextracellular matrix tissue.

Suitably, extracellular matrix is treated to reduce the GAG content.Preferably, the extracellular matrix is treated to reduce the GAGcontent after the extracellular matrix is micronized.

Typically, the porous scaffold is cross-linked.

Preferably, the extracellular matrix is hyaline cartilage (preferablyarticular cartilage) ECM or growth plate ECM.

Preferably, the extracellular matrix is decellularised before or aftermicronizing, ideally after micronisation.

Suitably, the method of the invention includes an additional step ofseeding the scaffold with a biological material, for example cells,preferably mesenchymal cells, or a biological molecule, for example agrowth factor. This could be achieved by, for example, soaking theprepared scaffold in a solution containing the growth factor or cells ofinterest. Suitably, the biological material or molecule (biologic) isselected from the groups of: cells; and biological growth factors.Typically, the biological growth factors are selected from the groupconsisting of one or more of the TGF-β superfamily, (IFG, FGF, BMP,PDGF, EGF) or cannabinoids. These growth factors can also be includedduring the production process as opposed to post-fabrication soaking ofthe scaffolds.

In a preferred embodiment, the invention provides a method for making aporous devitalised scaffold suitable for use in repair of osseous,chondral, or osteochondral defects in a mammal, the method comprisingthe step of:

-   -   providing micronized extracellular matrix (ECM) tissue having a        mean particle size of 30-70 microns;    -   mixing the micronized extracellular matrix with a liquid to        provide a slurry comprising 200-300 mg/ml micronized ECM; and    -   freeze-drying the slurry to provide the porous scaffold.

The invention also relates to a method of making a multilayer scaffoldcomprising the steps of making a first layer comprising a porousscaffold according to a method of the invention, making a second layercomprising a porous scaffold according to a method of the invention,wherein the first layer is attached to the second layer.

Preferably, the process includes a step of attaching the first layer tothe second layer to form the multilayer scaffold.

In one embodiment, the first layer comprises ECM from a first source andthe second layer comprises ECM from a different source to the firstsource. Preferably, the first source of ECM is hyaline cartilage ECM andthe second source of ECM is growth plate ECM. The latter type ofmultilayer scaffolds are suitable for repair or treatment ofosteochondral defects.

Suitably, the layers are attached together by means of freeze-drying.Thus, for example, the layers may be freeze-dried independently, andthen placed in a mould and freeze-dried together. Alternatively, onelayer may be freeze-dried and then placed in a mould with a slurry andfreeze-dried to form the layered scaffold. Other methods of attachingthe two layers include use of adhesives, stitching and intermediatebonding layers.

In a preferred embodiment, the invention also to a method of making amultilayer scaffold comprising the steps of making a first layercomprising a porous scaffold according to a method of the invention inwhich the ECM is cartilage ECM, making a second layer comprising aporous scaffold according to a method of the invention in which the ECMis growth plate ECM, wherein the first layer is attached to the secondlayer

The invention also relates to a porous scaffold formed according to amethod of the invention.

The invention also relates to a porous multilayer scaffold formedaccording to a method of the invention.

The invention also provides a porous scaffold typically suitable for usein repair of osseous, chondral, or osteochondral defects in a mammal andcomprising a porous freeze-dried matrix formed from microniseddecellularised extracellular matrix or solubilised and crosslinkedextracellular matrix.

Preferably, the micronized extracellular matrix tissue has a meanparticle size of 10-200 microns, ideally 20-70 microns.

Typically, the micronized extracellular matrix tissue is cryomilledextracellular matrix tissue.

Suitably, extracellular matrix comprises reduced GAG content.

Typically, the porous scaffold is cross-linked.

Preferably, the extracellular matrix is hyaline cartilage ECM (ideallyarticular cartilage ECM) or growth plate ECM.

Preferably, the micronised extracellular matrix is decellularised.

Suitably, the porous scaffold is seeded with a biological material, forexample cells, preferably mesenchymal stem cells, or a biologicalmolecule. Preferably, the ECM is growth plate or hyaline cartilage ECM,and the porous scaffold is seeded with cells, preferably mesenchymalstem cells.

The invention also provides a porous scaffold according to the inventionsuitable for use in repair of chondral defects in a mammal, in which theextracellular matrix is hyaline (ideally articular) cartilageextracellular matrix.

The invention also provides a porous scaffold according to the inventionsuitable for use in repair of osseous defects in a mammal, in which theextracellular matrix is growth plate tissue extracellular matrix.

A multilayer scaffold typically suitable for use in repair ofosteochondral defects in a mammal and having a first layer comprising aporous scaffold of the invention and a second layer comprising a porousscaffold of the invention, in which the first layer is attached to thesecond layer.

In one embodiment, the first layer of porous scaffold comprises ECM froma first source and the second layer of porous scaffold comprises ECMfrom a different source to the first source. Preferably, the firstsource of ECM is hyaline cartilage ECM and the second source of ECM isgrowth plate ECM. The latter type of multilayer scaffolds are suitablefor repair or treatment of osteochondral defects.

Suitably, the layers are seamlessly attached by means of, for example,freeze-drying. Thus, for example, the layers may be freeze-driedindependently, and then placed in a mould and freeze-dried together.Alternatively, one layer may be freeze-dried and then placed in a mouldwith a slurry and freeze-dried to form the layered scaffold. Othermethods of attaching the two layers include use of adhesives, stitchingand intermediate bonding layers.

The invention also relates to a porous scaffold or multilayer scaffoldof the invention for use in a method of treating osseous, chondral, orosteochondral defects in a mammal, in which the porous scaffold isinserted into the defect.

The invention also relates to a porous scaffold of the invention for usein a method of treating chondral defects in a mammal, in which theporous scaffold comprises cartilage ECM and in which the porous scaffoldis inserted into the chondral defect.

The invention also relates to a porous scaffold of the invention for usein treating osseous defects in a mammal, in which the porous scaffoldcomprises growth plate ECM and in which the porous scaffold is insertedinto the osseous defect. The porous scaffold comprises cells, ideallymesenchymal stem cells, although the scaffold may also be cell-free.

The invention also relates to a multilayer scaffold of the invention foruse in treating osteochondral defects in a mammal, in which themultilayer scaffold comprises a first layer of porous scaffoldcomprising hyaline cartilage ECM and a second layer of porous scaffoldcomprising growth plate ECM, and in which the multilayer scaffold isinserted into the osteochondral defect. The first and/or second layer ofporous scaffold may comprise cells, ideally mesenchymal stem cells,although the scaffold is preferably cell-free.

In another aspect, the invention relates to a gel suitable for use inrepairing osseous, chondral, or osteochondral defects in a mammal andcomprising a gel base and micronized ECM homogenously distributedthroughout the gel base.

Typically, the gel base comprises fibrin.

Ideally, the gel is injectable.

The invention also relates to a method of making a gel suitable for usein repairing osseous, chondral, or osteochondral defects in a mammal andcomprising the steps of mixing micronised ECM with a gel base.Typically, the process comprises a step of mixing micronised ECM with aliquid gel base precursor, and then adding to the mixture an activatorcapable of converting the liquid gel base precursor to a gel base.Suitably, the gel base precursor is fibrinogen, the activator isthrombin.

The invention also relates to a method of making a porous devitalisedECM-based scaffold comprising the step of mixing ECM-producing cellswithin a hydrogel base, culturing the mixture in-vitro such that theECM-producing cells deposit ECM within the mixture, and thenfreeze-drying the mixture to provide the porous devitalised ECM-basedscaffold.

Typically, the ECM-producing cells are selected from chondrocytes andosteoblasts.

The invention also relates to a porous ECM-based scaffold formedaccording to the method of the invention.

The invention also relates to a porous ECM-based scaffold formedaccording to the method of the invention in a micronized form.

The invention also relates to micronized growth plate ECM, a slurrycomprising micronized ECM, or a freeze-dried scaffold formed from aslurry of micronized growth plate ECM.

The invention also relates to a porous scaffold formed from micronized,freeze-dried, growth plate ECM.

The invention also relates to a porous freeze-dried scaffold comprisingcryomilled ECM.

The invention also relates to a porous scaffold formed from solubilisedand crosslinked, freeze-dried, growth plate ECM.

The invention also relates to a porous multilayer scaffold comprising atleast first and second layers, the first layer comprising a porousfreeze-dried scaffold formed from micronized hyaline cartilage ECM andthe second layer comprising a porous freeze-dried scaffold formed frommicronized growth plate cartilage ECM.

The invention also relates to a porous growth plate ECM-based scaffoldformed according to the method of the invention in a solubilised form.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Concentration modulates scaffold morphology. Helium ion (HIM)micrographs showed different architecture in scaffolds when cartilageECM slurry concentration was altered: (A) 250 mg/ml; (B) 500 mg/ml; (C)1000 mg/ml. Mean pore size decreased with increased concentration ofECM.

FIG. 2: Distribution of live cells. Confocal microscopy at day 1 and day28 of human infrapatellar fat pad derived stem cells seeded in ECMderived scaffolds: (A) 250 mg/ml, (B) 500 mg/ml and 1000 mg/ml. Picturerepresents cross-section of ECM derived scaffolds. Poor cellularpenetration at day 1 was observed in the (B) 500 mg/ml and (C) 1000mg/ml scaffolds, which contrasts with the 250 mg/ml scaffold where ahomogeneous cellular infiltration was observed (A). At day 28, scaffoldwith 1000 mg/ml of ECM continued to show poor stem cells infiltration(F).

FIG. 3: sGAG deposition for day 0, 7, 14 and 28. Histological images ofglycosaminoglycans (GAG) (alcian blue) and cell nuclei (nuclear fastred) staining for ECM derived scaffolds—250, 500 and 1000 mg/ml—at day0, 7, 14 and 28 of culture (A). In (A) it is possible to observe strongGAG deposition and cell distribution for the 250 mg/ml scaffold. Withhigh magnification it is possible to observe the superior GAG depositionfor 250 (B), followed by 500 (C) and 1000 mg/ml scaffold (D).

FIG. 4: TGF-β3 release profile for constructs with or without EDACcrosslinking. ELISA results for TGF-β3 release into the media fromTGF-β3 loaded ECM derived scaffold indicates slower release rate forscaffolds with EDAC crosslinking, with significant difference at day 4(n=6, *p<0.05).

FIG. 5: Tailored sGAG scaffold morphology. Helium ion (HIM) micrographsshowed altered pore size and architecture in tailoring GAG concentrationof scaffolds: (A) 5% GAG; (B) 50% GAG; (C) 100% GAG. Mean pore sizeincreased with decreasing GAG concentration.

FIG. 6: sGAG Histology. GAG staining on decellularized tailored GAGcartilage explants. micrographs showed in tailoring GAG concentration:(A) 5% GAG; (B) 50% GAG; (C) 100% GAG.

FIG. 7: TGF-β3 release profile from tailored GAG cartilage ECMscaffolds. ELISA results for TGF-β3 release into the media from TGF-β3loaded ECM derived scaffold 4 (n=4). By removing sGAGs from the ECMprior to scaffold fabrication, it is possible to slow the release ofgrowth factors from the construct.

FIG. 8: Biochemical assay for DNA and GAG content of tailored GAGscaffold.

FIG. 9: Biochemical assays performed on cartilage tissues engineered invitro using tailored GAG scaffolds seeded with human stem cells

FIG. 10: Histological staining for sGAG (Alcian blue) and Collagen(Picrosirius red) of cartilage tissues engineered in vitro using ECMderived scaffolds seeded with human stem cells.

FIG. 11: Gross appearance of Fibrin-ECM particle constructspost-gelation

FIG. 12: TGF-β3 release profile for Fibrin hydrogel loaded with ECMparticles. ELISA results for TGF-β3 release into the media from TGF-β3loaded ECM derived particles (n=4).

FIG. 13: GAG content of cartilage tissues engineered in vitro using ECMparticle loaded hydrogels. Fibrin hydrogels containing ECM particlesloaded with TGF-β3 showed higher GAG accumulation than constructs whereTGF-β3 was either added directly to the media or added to controlgelatine microparticles (MPs).

FIG. 14: Histology. GAG and Collagen staining of cartilage tissuesengineered in vitro within fibrin hydrogels loaded with ECM derivedparticles

FIG. 15: Gross morphology after of tissues generated in vivo usingproposed injectable construct.

FIG. 16: Histology. GAG and Collagen staining for tissues generated invivo.

FIG. 17. A representative PDMS mould used to control freeze-drying ofgrowth plate ECM to specific dimensions

FIG. 18: Schematic outlining the steps involved in endochondralossification, whereby a cartilage template is converted to a mature bone

FIG. 19: Histological analysis of constructs at day 0 and following 28days of culture in either chondrogenic or osteogenic medium,demonstrating the deposition of the main constituents of cartilage, sGAGand collagen.

FIG. 20: MSC-seeded growth plate ECM constructs with positive collagentype II and collagen type X staining

FIG. 21: Mineral deposition was observed in MSC-seeded growth plate ECMscaffolds cultured in either chondrogenic or osteogenic medium, incomparison to MSCs seeded on a cartilage ECM construct which onlymineralised in osteogenic culture conditions.

FIG. 22: An image of a bi-layered construct containing cartilage ECM inthe top layer and growth plate ECM in the bottom layer and histologicalanalysis of tissue deposited by MSCs seeded onto osteochondral scaffoldsafter 28 days in culture.

FIG. 23: Reconstructed images of growth plate scaffold treated anduntreated cranial defects at 4 and 8 weeks showing the level ofmineralisation achieved

FIG. 24: Reconstructed images of growth plate scaffold treated anduntreated femoral defects at 4 and 8 weeks showing the level ofmineralisation achieved

FIG. 25: Histological analysis of repair tissue formed across thecranial defect after 4 and 8 weeks either (a) untreated or (b) treatedwith the growth plate scaffold. (c) Higher power images of the repairtissue, demonstrating de novo bone forming both upon and within theoriginal growth plate ECM tissue.

FIG. 26: Biochemical assays for DNA and GAG content of solubilised ECMscaffold (a). Scaffolds were generated using micronized ECM (High GAG)or solubilised ECM. Macroscopic images of wet and dry solubilised ECMscaffolds (b).

FIG. 27: Biochemical assays performed on cartilage tissues engineered invitro using solubilised or micronized ECM scaffolds seeded with humanstem cells (a). Scaffolds were generated using micronized ECM (High GAG)or solubilised ECM. Gross morphology of tissues generated usingscaffolds (b).

DETAILED DESCRIPTION OF THE INVENTION Definitions

In this specification, the term “porous” as applied to a scaffold shouldbe understood to mean having a porosity of at least 90% as determinedusing the method of Gleeson et al (J. P. Gleeson, N. A. Plunkett, F. J.O'Brien—Addition of hydroxyapatite improves stiffness, interconnectivityand osteogenic potential of a highly porous collagen-based scaffold forbone tissue regeneration—Eur Cell Mater, 20 (2010), pp. 218-223) In oneembodiment, the scaffold (or each layer in the scaffold) has a porosityof at least 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%. Ideally, thescaffold has a porosity of at least 98%, ideally at least 98.5%.

In this specification, the term “osseous defect” should be understood tomean any defect within bony tissue.

In this specification, the term “chondral defect” should be understoodto mean any defect within the articular surface of a joint that does notpenetrate through the subchondral bone.

In this specification, the term “osteochondral defect” should beunderstood to mean a defect to the articular surface that affects boththe articular cartilage and the underlying bone.

In this specification, the term “extracellular matrix tissue” or“extracellular matrix” or “ECM” should be understood to mean acollection of extracellular molecules secreted by cells that providesstructural and biochemical support to the surrounding cells. The ECM maybe obtained from a mammal, for example a human or a non-human mammal, orit may be engineered in-vitro using published techniques, for exampleVinardell et al (Vinardell, T., Sheehy, E., Buckley, C. T., Kelly, D. J.A comparison of the functionality and in vivo phenotypic stability ofcartilaginous tissues engineered from different stem cells sources.Tissue Engineering Part A, 18(11-12), 1161-1170, 2012) and Buckley et al(Buckley, C. T., Vinardell, T., Kelly, D. J. Oxygen TensionDifferentially Regulates the Functional Properties of CartilaginousTissues Engineered from Infrapatellar Fat Pad Derived MSCs and ArticularChondrocytes. Osteoarthritis and Cartilage, 18 (10), 1345-1354, 2010).Examples of extracellular matrix for the purpose of the presentinvention include cartilage ECM (obtained from porcine articularcartilage tissue) and growth plate ECM (typically obtained from theepiphysial plate of porcine tibia or femora).

In this specification, the term “hyaline cartilage ECM” should beunderstood to mean ECM obtained from hyaline cartilage which is a tissuefound, for example, in the ear and nose and on joint surfaces. It ismostly composed of type II collagen and chondroitin sulphate.

In this specification, the term “articular cartilage ECM” should beunderstood to mean ECM obtained from articular cartilage, which is aform of hyaline cartilage found at the articular end of joints.

In this specification, the term “growth plate ECM” or “growth platetissue ECM” should be understood to mean ECM obtained from growth platetissue of developing bones, typically developing long bones. This couldinclude the epiphyseal plate in the metaphysis of a long bone, orarticular cartilage from skeletally immature joints as this tissue isalso known to act as a surface growth plate during development andskeletal maturation.

In this specification, the term “micronised” as applied to ECM should beunderstood to mean provided in a particulate form, in which theparticles of ECM have a mean particle size of less than 200 microns asdetermined using routine light microscopy. Preferably, the micronisedECM has a mean particle size of less than 150 or 100 microns. Ideally,the micronized ECM has a mean particle size between 20 and 200 microns,20 and 150 microns, 20 and 100 microns, 20 and 70 microns, 30 and 70microns, 30 and 60 microns, 40 and 60 microns, and ideally about 50microns. Methods of micronisation include milling, cryomilling,

In this specification, the term “cryomilled” should be understood tomean a process in which a material is cryogenically frozen and thenmilled. Examples of cryomilling machines include the RETCH CRYOMILL™.

In this specification, the term “solubilised” should be understood tomean a process by which ECM tissue is digested, ideally enzymaticallydigested, to become soluble in an aqueous solvent. Suitably solubilisingagents will be known to the person skilled in the art, and includeenzymes and denaturing agents such as urea. An example of an enzyme thatcan be used to digest ECM tissue to become soluble is a protease, forexample pepsin, or a collagense. Preferably, the solubilised ECM will bea purified collagen with substantial removal of GAG and xenogeneic DNA.Ideally, the solubilised ECM will have greater than 50%, 60%, 70%, 80%or 90% removal of GAG and DNA when compared to native ECM tissue.

In this specification, the term “freeze-drying” as applied to a slurryrefers to a process in which the slurry is frozen, typically to a finalfreezing temperature of from −10° C. to −70° C. and then sublimatedunder pressure. In one embodiment, the desired final freezingtemperature is between −10° C. and −70° C. Suitably, the desired finalfreezing temperature is between −30° C. and −50° C. Typically, thedesired final freezing temperature is between −35° C. and −45° C.,ideally about −40° C. In one embodiment of the invention, freezing orfreeze-drying is carried out at a constant cooling rate. This means thatthe rate of cooling does not vary by more than +/−10% of the targetcooling rate, i.e. if the desired rate of cooling is 1.0° C./min, andthe actual rate of cooling varied between 0.9° C./min and 1.1° C./min,this would nonetheless still be considered to be a constant coolingrate. Typically, the constant cooling rate is between 0.1° C./min to 10°C./min. Preferably, freeze-drying is carried out at a constant coolingrate of between 0.5° C./min to 1.5° C./min. More preferably, freezing orfreeze-drying is carried out at a constant cooling rate of between 0.8°C./min to 1.1° C./min. Typically, freezing or freeze-drying is carriedat a constant cooling rate of about 0.9° C./min. The temperature of thefreeze-drying chamber at a start of the freeze-drying process (i.e. whenthe slurry is placed in the chamber) is usually greater than 0° C.,preferably at about ambient temperature. The sublimation step isgenerally carried out after the final freezing temperature is reached.This step involves heating the freeze-drying chamber to a sublimationtemperature (generally about 0° C.), preferably at a constant heatingrate. The process typically includes a final sublimation step where anice phase in the formed scaffold is sublimated under vacuum for asuitable period of time.

In this specification, the term “slurry” should be understood to mean asuspension of micronized ECM in a solvent, suitably an aqueous solvent,for example water. Typically, the slurry comprises less than 500, 400,300 mg/ml micronized ECM. Suitably, the slurry comprises 100-500,100-400, 200-300, 230-270, and ideally about 250 mg/ml micronized ECM.

In this specification, the term “cross-linked” should be understood tomean treated to introduce cross-links between different polymericmolecules in the ECM. The ECM may be micronised ECM or solubilised ECM.Crosslinking may be performed on the solubilised ECM or on the formedfreeze-dried scaffold. Typically, the scaffold is cross-linked by one ormore of the means selected from the group comprising: dehydrothermal(DHT) cross-linking; and chemical cross-linking. When crosslinking is beperformed on the solubilised ECM, the crosslinking agent is typically achemically crosslinking agent. Suitable chemical cross-linking agentsand methods will be well known to those skilled in the art and include aglyoxal, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride(EDAC) or Glutaraldehyde. Ideally, the scaffold is cross-linked usingDHT and EDAC cross-linking. Cross-linking can be carried out at anystage of the fabrication process. In a preferred embodiment, scaffoldpore symmetry can be controlled by varying the degree of cross-linkingwithin each respective layer using cross linking methods familiar to oneskilled in the art. Similarly, in another embodiment, scaffoldpermeability or flow conductivity can be varied by varying themechanical properties of the scaffold using either cross linking orother stiffness improvement methodologies known to one skilled in theart.

In this specification, the term “GAG” should be understood to meanglycosaminoglycan, particularly sulphated glycosaminoglycans.

In this specification, the term “reduced GAG content” as applied to ECMfrom a given source should be understood to mean a GAG content that isreduced compared to natural ECM from the same source, for example lessthan 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5% GAG content ofnatural ECM. Methods of reducing GAG content include the use of buffers,detergents (such as Sodium dodecyl sulfate or Triton-X or Sodiumdeoxycholate) or other chemicals (e.g. chondroitinase ABC) known toreduce the sGAG content of tissues.

In this specification, the term “decellularised” or “devitalised” as aapplied to a material (for example ECM, a scaffold, or a gel) should beunderstood to mean that the cellular content of the material is reducedpartially or preferably completely. Method of decellularising a materialinclude chemical nucleic acid digestion, possibly following partial ortotal removal of matrix components from the ECM.

In this specification, the term “seeding” as applied to a scaffoldshould be understood to mean incorporating a biological material into ascaffold. Method of seeding a scaffold include soaking the scaffold in asolution containing the biological material for a sufficient time toallow the biological material infiltrate the pores of the scaffold.

In this specification, the term “cells” should be understood to mean anytype of cell, particularly stem cells, chondrocytes, and osteoblasts.Preferably, the cells are mesenchymal stem cells.

In this specification, the term “biological material” should beunderstood to mean proteins, peptides, nucleic acid, nucleic acidconstructs, nucleic acid vectors, or chemical molecules havingbiological activity. Preferably, the biological material comprises abiological growth factor, for example one or more of the TGF-βsuperfamily, (IFG, FGF, BMP, PDGF, EGF) or cannabinoids.

In this specification, the term “cannabinoids” should be understood tomean a biological compound which can be naturally or syntheticallyderived and that acts on the cannabinoid receptor types 1 and/or 2 (CB₁and CB₂), for example Δ9-tetrahydrocannabinol (Δ9-THC).

In this specification, the term “gel base” should be understood to meana matrix having both solid and liquid properties. An exemplary gel baseis an agarose gel.

In this specification, the term “injectable” should be understood tomean that the gel is sufficiently deformable to enable it to be injectedinto a defect in cartilage or bone.

EXPERIMENTAL

Development of Decellularized ECM Derived Scaffolds with a Uniform PoreSize.

Cartilage used in the fabrication of ECM derived scaffolds washarvested, in sterile conditions, from the femoral condyles of femalepigs (3 months old) shortly after sacrifice. The cartilage was firstbroken up into small pieces using a scalpel. Cartilage particles werethen broken up using a cryogenic mill (6770 Freezer/Mill, SPEX, UK).These small pieces of cartilage where then homogenized in distilledwater (dH2O) using a homogenizer (IKAT10, IKA Works Inc, NC, USA) tocreate a cartilage slurry (250 mg/ml). The slurry was transferred tocustom made moulds (containing wells 5 mm in diameter and 3 mm inheight) and freeze-dried (FreeZone Triad, Labconco, KC, USA) to produceporous scaffolds. Briefly, the slurry was frozen to −30° C. (1° C./min)and kept at that temperature for one hour. The temperature was thenincreased to −10° C. (1° C./min), followed by a hold of 24 hours andthen finally increased to room temperature (0.5° C./min). Next, twodifferent crosslinking techniques were applied to the scaffolds. Thescaffolds underwent DHT and 1-Ethyl-3-3dimethyl aminopropyl carbodiimide(EDAC) crosslinking The DHT process was performed in a vacuum oven(VD23, Binder, Germany), at 115° C., in 2 mbar for 24 hours. The EDAC(Sigma-Aldrich, Germany) crosslinking consisted of chemical exposure for2 hours at a concentration of 6 mM in the presence ofN-Hydroxysuccinimide (NHS) (Sigma-Aldrich, Germany). A molar ratio of2.5 M EDAC/M N-Hydroxysuccinimide was used. After EDAC crosslinking thescaffolds were washed twice in sterile PBS (Sigma-Aldrich, Germany).

Development of Decellularized ECM Derived Scaffolds with Controlled PoreSize and Tailored Growth Factor Release Rates.

Articular cartilage was harvested from femoral condyles of female 4months old pigs under sterile condition shortly after sacrifice. Allsteps of the decellularization and tailoring GAG protocol were performedin 2 mL working volume at room temperature. This protocol consists ofthree phases. In Phase I, the 50 and 5% GAG groups were incubated inbasic buffer (10 mM Tris-HCl (pH 8.0)) containing 100 mM DTT, 2 mMMgCl2, and 10 mM KCl for 24 hrs; and anatomically adjacent pieces ofcartilage subjected to 1 min incubations for 100% GAG group. The 5% GAGgroups were additionally subjected to 0.5% SDS treatment with basicbuffer containing 100 mM DTT, 2 mM MgCl2, and 10 mM KCl for 24 hrs.Following sGAG removal, nucleic acid digestion (2.5 Kunitz units/mLdeoxyribonuclease I, 7.5 Kunitz units/mL ribonuclease A, 0.15 M NaCl, 2mM MgCl₂ (H2O) in 10 mM Tris-HCl (pH 7.6)) was performed for 24 h andwashout (10 mM Tris-buffered saline (pH 7.5)) for 48 h. In phase II, thecartilage tailored GAG-ECM scaffolds were prepared by cryo-millingfollowed by DHT+EDAC crosslinking as described in section 1 above.

Development of Solubilised ECM Derived Scaffolds

Cartilage used in the fabrication of ECM derived scaffolds washarvested, in sterile conditions, from the femoral condyles or growthplates of female pigs (3 months old) shortly after sacrifice. Thecartilage was first broken up into small pieces using a scalpel. ECMtissue was then transfer to sterile containers. ECM tissue was thenpre-treated with 0.2M NaOH for 24 hours at 4° C. After washing andremoval of pre-treatment solution, the ECM tissue was then digested withpepsin in 0.5 M Acetic Acid. Pepsin is added at a concentration of ˜1500units pepsin per 50 mg ECM tissue. The ECM was then incubated in thepepsin solution for 24 hours at <20° C. with rotation at a speed of 4rpm. Salt precipitation was then performed to extract purified collagenusing concentration of NaCl between 0.1M-5M. In order to remove anyremaining salt, acid or pepsin, dialysis can be performed on thesolubilised collagen. Dialysis was performed against 0.02 M Na₂HPO₄ (pH9.4) for 24 h at 4° C. The solubilised collagen can then befreeze-dried. To generate scaffolds, the freeze dried collagen wasrehydrated in an aqueous solution at a concentration range of 1 mg/ml to200 mg/ml preferably, 20 mg/ml. Once rehydrated the collagen can then becross-linked to form a gel with Glyoxal at a concentration between 1 mMand 50 mM preferably, 10 mM. The solution is then incubated for 30minutes at 37° C. to allow cross-linking to take place. After incubationthe gel can then be transferred to moulds and freeze-dried to createscaffolds.

Development of Injectable Decellularized ECM Derived Particles as GrowthFactor Delivery Systems.

Particulated cartilage ECM is fabricated as described in 1 or 2 above.Instead of freeze-drying these particles to produce a porous scaffold,it is also possible to combine these particles with a hydrogel todevelop an injectable chondroinductive composite biomaterial that alsoacts as a growth factor delivery system.

One manifestation of this invention would be to combine ECM particleswith a fibrin hydrogel. The particulated cartilaginous material isincorporated into the hydrogel by mixing directly with the fibrinogen,with the desired ratio. Gelation occurs by adding thrombin to thefibrinogen/ECM-particles slurry. Appropriate mixing ensures ahomogeneous distribution of bioactive cartilage ECM-derivedmicro-particles within the hydrogel.

Development of Decellularized Growth Plate ECM Derived Scaffolds.

Growth plate used in the fabrication of ECM derived scaffolds washarvested, in sterile conditions, from the femur, fibula and tibia offemale pigs (3 months old) shortly after sacrifice. The growth plate wasfirst broken up into small pieces using a scalpel, and then broken upusing a cryogenic mill (6770 Freezer/Mill, SPEX, UK). These small piecesof growth plate were then homogenized in distilled water (dH₂O) using ahomogenizer (IKAT10, IKA Works Inc, NC, USA) to create a slurry (250mg/ml). The slurry was transferred to custom made moulds andfreeze-dried (FreeZone Triad, Labconco, KC, USA) to produce porousscaffolds. Briefly, the slurry was frozen to −30° C. (1° C./min) andkept at that temperature for one hour. The temperature was thenincreased to −10° C. (1° C./min), followed by a hold of 24 hours andthen finally increased to room temperature (0.5° C./min). Next, twodifferent crosslinking techniques were applied to the scaffolds. Thescaffolds underwent DHT and 1-Ethyl-3-3dimethyl aminopropyl carbodiimide(EDAC) crosslinking. The DHT process was performed in a vacuum oven(VD23, Binder, Germany), at 115° C., in 2 mbar for 24 hours. The EDAC(Sigma-Aldrich, Germany) crosslinking consisted of chemical exposure for2 hours at a concentration of 6 mM in the presence ofN-Hydroxysuccinimide (NHS) (Sigma-Aldrich, Germany). A molar ratio of2.5 M EDAC/M N-Hydroxysuccinimide was used. After EDAC crosslinking thescaffolds were washed twice in sterile PBS (Sigma-Aldrich, Germany).

Results obtained from both in vitro and in vivo characterisation of thegrowth plate scaffold will be presented below, and demonstrate itspotential for use in bone tissue regeneration. Also, we will display theability of the growth plate scaffold layer to be combined with acartilage ECM layer to generate an osteochondral graft which can bepotentially applied to repair both bone (osteo) and cartilage (chondral)layers simultaneously.

The invention is not limited to the embodiments hereinbefore describedwhich may be varied in construction and detail without departing fromthe spirit of the invention.

1.-65. (canceled)
 66. A method for making a porous scaffold suitable foruse in repair of osseous, chondral, or osteochondral defects in amammal, the method comprising the step of: providing micronizedextracellular matrix (ECM) tissue; mixing the micronized extracellularmatrix with a liquid to provide a slurry; and freeze-drying the slurryto provide the porous scaffold, wherein the extracellular matrix istreated to reduce the GAG content to less than 90% of the GAG content ofuntreated ECM, and wherein the ECM tissue is cartilage ECM.
 67. A methodaccording to claim 66 in which the micronized cartilage extracellularmatrix tissue has a mean particle size of 10-200 microns.
 68. A methodaccording to claim 66 in which the slurry comprises 100-400 mg/mlmicronized cartilage ECM tissue.
 69. A method according to claim 66 inwhich the micronized cartilage extracellular matrix tissue is cryomilledcartilage extracellular matrix tissue.
 70. A method according to claim66 in which the porous scaffold is cross-linked.
 71. A method accordingto claim 66 in which the cartilage extracellular matrix is hyalinecartilage ECM or growth plate ECM.
 72. A method according to claim 66 inwhich the cartilage extracellular matrix is decellularised before orafter micronizing.
 73. A method according to claim 66 in which themethod of the invention includes an additional step of seeding thescaffold with a biological material selected from cells or a biologicalgrowth factor.
 74. A method according to claim 73 in which: the cellsare selected from the group consisting of stem cells, chondrocytes,mesenchymal cells and osteoblasts; and/or in which the biological growthfactor is selected from the group consisting of one or more of the TGF-βsuperfamily or cannabinoids.
 75. A method of making a multilayerscaffold comprising the steps of making a first layer comprising aporous scaffold according to a method of claim 66, making a second layercomprising a porous scaffold according to a method of claim 66, whereinthe first layer is attached to the second layer.
 76. A method accordingto claim 75 in which the process includes a step of attaching the firstlayer to the second layer to form the multilayer scaffold, in which thefirst layer comprises hyaline cartilage ECM and the second layercomprises growth plate ECM.
 77. A porous scaffold suitable for use inrepair of osseous, chondral, or osteochondral defects in a mammal andcomprising a porous freeze-dried matrix formed from microniseddecellularised extracellular matrix tissue, wherein the extracellularmatrix tissue comprises less than 90% of the GAG content of natural ECM,and wherein the extracellular matrix tissue is cartilage extracellularmatrix tissue.
 78. A porous scaffold according to claim 77 in which theporous scaffold is cross-linked.
 79. A porous scaffold according toclaim 77 in which the cartilage extracellular matrix is hyalinecartilage extracellular matrix or growth plate extracellular matrix. 80.A multilayer scaffold suitable for repair of osteochondral defects in amammal and having a first layer comprising a porous scaffold accordingto claim 77 in which the cartilage extracellular matrix is hyalinecartilage extracellular matrix and a second layer comprising a porousscaffold according to claim 77 in which the cartilage extracellularmatrix is growth plate extracellular matrix, in which the first layer isattached to the second layer.